Nucleus pulpous (NP) tissues were collected from 110 patients with intervertebral disc degeneration (IDD) who underwent lumbar microdiscectomy (mean age 56.3 ± 9.1 years). All IDD patients were clinically diagnosed by two qualified spine surgeons via physical examination and MRI. Based on their Pfirrmann classification, all patients were classified as mild/moderate degeneration (grades 1–2) or serious degeneration (grades 3 or 4). The normal NP tissues were collected from 103 patients with lumbar vertebral fracture who underwent anterior spine surgery (mean age 53.7 ± 4.6 years). These trauma patients had no prior history of preoperative low back pain or IDD.
This study was approved by the Ethics Committee of The First Affiliated Hospital of Guangxi Medical University (2018-KY-NSFC-025). All patients involved in the study gave signed consent for the use of their information and clinical condition in this study.
The injury-induced IDD model and therapeutic experiment
The injury-induced IDD mice model was established via needle puncture as previously described.[23, 36] Briefly, following general anesthesia of 12-week-old C57BL/6 mice, their coccygeal discs Co6/Co7 were exposed and then punctured with 31G syringe needle through the AF to NP. The needle was then left in the intervertebral disc at a depth of 1.5 mm for period of 10 seconds. The adjacent Co7/Co8 disc levels were not punctured as they were used as contrast segments.
For the therapeutic tests, 20 male mice that underwent the previously described injury-induced IDD surgery were randomly divided into 4 groups (n = 5): group 1 (agomir-control); group 2 (agomir-338-3p); group 3 (antagomir-control); and group 4 (antagomir-338-3p) (treatment group). The agomir- or antagomir-338-3p and their controls were acquired from RiboBio (RiboBio Co., Guangzhou, China). The injection mice with agomir-338-3p/agomir-control or antagomir-338-3p/antagomir- control were performed on the 1st, 7th, and 14th days post-needle puncture intervention. The disc samples were then harvested at two different time-periods: 6th week and 12th week post intervention.
The ethical committee of The First Affiliated Hospital of Guangxi Medical University approved the animal experimentation protocol, and all animal experimentations were conducted in accordance with the approved guidelines.
The human NP tissues were sliced into 1 mm3 pieces and washed twice with 10% fetal bovine serum (PBS) (GIBCO, NY, USA), followed by incubation in DMEM (GIBCO, NY, USA). Subsequently, the resuspension of NP cells were cultured in DMEM, 1% penicillin-streptomycin and 10% FBS. The cells were then cultured in an incubator at 37 °C and 5% CO2, and then dissociated with trypsin and split at a ratio of one to three.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Trizol (Invitrogen Life Technologies, CA, USA) was used for total RNAs extraction. The RNA templates were then synthesized into cDNA using the iScripts cDNA Synthesis kit (Quanta Biosciences, MD, USA), and GAPDH used as control for normalization. The isolated miRNA were then quantified using qScript microRNA cDNA Synthesis Kit (Quanta BioSciences, MD, USA), with U6 snRNA used as the internal control. Thereafter, SYBR Green real-time PCR kit (Quanta Biosciences) was then used to perform the qRT-PCR, with comparative threshold cycle (△△Ct) adopted for the calculation of gene expression. The specific primers are listed in Supplementary Table 2.
miRNA microarrays and miRNA target prediction
The miRNA microarray was conducted using Affymetrix® 4.0 miRNA Array (USA). Total RNAs were obtained from six individual NP samples with or without IDD. Based on volcano plot and fold change filtering, we identified the differentially expressed miRNAs in IDD. Candidate miRNAs were identified if they met the following criteria: (1) exhibited more than 5 fold changes or less than 0.2 fold changes; (2) p-values less than 0.05. Gene Cluster software (Stanford University) and DAVID software were used for hierarchical cluster analysis and functional group analysis respectively. TargetScanHuman (www.targetscan.org/) and microRNA.org were used to forecast the miRNA target genes and to analyze mRNA binding sites.
miR-338-3p and SIRT6 transfection
For overexpression or silence of miR-338-3p, human NP cells were transfected with miR-338-3p mimics or inhibitors or their negative controls (Cat. No: 4464061 and 4464079, Life Technologies) using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). For the suppression of SIRT6 expression, transient transfection of human NP cells with SIRT6 siRNA or control siRNA (Cat. No: 116148 and 4459408, Thermo Fisher Scientific) were carried out using Lipofectamine 3000 Transfection Reagent (Invitrogen). The SIRT6 expression plasmid (pcDNA3.1(+)/SIRT6) was obtained from Invitrogen.
Luciferase constructs and reporter assay
SIRT6 mRNA 3’UTR fragments including the binding sites of wild-type (wt) and mutant (mut) miR-338-3p, were inserted into the psiCHECK-2 luciferase reporter vector (Promega, WI, USA). A miR-338-3p expression plasmid or a control plasmid was then co-transfected with SIRT6 wt- or mut-3’UTR and their respective control plasmids into human NP cells. All Luciferase assays were conducted using Dual-Glo Luciferase Assay System (Promega, WI, USA), and luciferase activity measured by a fluorescence microplate reader (BioTek, USA).
Flow cytometry, Cell Counting Kit-8 (CCK8) and EdU assay
Cellular apoptosis was measured using apoptosis kit with Annexin V-FITC/PI (Cat. No: V13242, Invitrogen), and analysis performed with Beckman Coulter EPICS Altra (USA). The detection of cellular proliferation was performed using CCK8 (Dojindo Laboratories, Kumamoto, Japan). Human NP cells that received different transfections (miR-338-3p mimics, control mimics, miR-338-3p inhibitor or control inhibitor) were then collected into 96-well plates, and then cultured for 24, 48, and 72 h. Afterwards, CCK8 was added to incubate the cultured cells for 3 h, with absorption evaluated at 450 nm. The degree of cellular proliferation was then assessed using EdU assay. The human NP cells were incubated with EdU medium (Sigma-Aldrich, MO, USA), and the collected cells stained with Hoechst 33258.
Fluorescence in situ hybridization (FISH)
For the analysis of human NP using FISH, digoxin-labeled miR-338-3p probes with locked nucleic acid modifications were designed and synthesized using Exiqon- QIAGEN (Hilden, Germany). The fluorescence signals were detected using the FISH Kit (Exiqon-QIAGEN), with Nikon A1Si Laser-Scanning Confocal Microscope (Nikon, Tokyo, Japan) used for image analysis.
Western blotting and co-immunoprecipitation
The proteins extracted from human NP cells using Micro BCA Protein Assay Kit (Cat. No: 23235, Thermo Fisher Scientific) were separated into equal amounts using 10% SDS/PAGE (sodium dodecyl sulfate-polyacrylamide gels), and then electroblotted onto a polyvinylidene fluoride membranes (Bio-Rad Laboratories). The membranes were then incubated with primary antibodies as follows: anti-Col II, anti-Aggrecan, anti-ADAMTS4, anti-MMP9, anti-Caspase-3, anti-Bax, anti-P16, anti-p-ERK1/2, anti-ERK1/2, anti-GRB2, anti-SIRT6, and anti-GAPDH (ab34712, ab36861, ab185722, ab38898, ab13847, ab132503, ab151303, ab214362, ab184699, ab32111, ab191385, and ab9485; Abcam, Cambridge, UK). After washing, the membranes were then treated with 1/2000 secondary goat anti-rabbit antibody (Abcam, Cambridge, UK). The resulting patterns were then interpreted using the Quantity One version 4.50 software (Bio-Rad, Hercules, CA, USA). The Co-immunoprecipitation analysis was performed on basis of nuclear and cytoplasmic proteins. Extraction Kit (Thermo Scientific, 78833) was then used to extract nuclear and cytoplasmic proteins from the human NP cells, and the expression levels of GRB2 and SIRT6 detected using anti-GRB2 (ab32111, Abcam) and anti-SIRT6 (ab191385, Abcam) antibodies.
Immunofluorescence and TUNEL staining
Intervertebral discs were fixed, decalcified and embedded in 10% formalin, 10% EDTA, and paraffin, respectively. Sagittal sections of disc tissue were then cut at every 3 µm from the midsagittal plane. The sections were then stained with alcian blue for histological analysis. Masuda’s method was then used for the grading score of the histological staining. The cultured NP cells were then fixed, permeabilized and blocked with 4% paraformaldehyde, 0.3% Triton X-100, and 1% BSA, respectively. For the immunofluorescence analysis, the following primary antibodies were incubated with sections of cells: anti-Mmp9, anti-Col II antibody (ab73734, ab34712; Abcam, Cambridge, UK) at 4 °C overnight. The sections and cells were then treated with a secondary antibody for 20 min at room temperature, followed by the capturing of immunofluorescence images using a Zeiss LSM780 confocal microscope (CarlZeiss, Oberkochen, Germany). For the detection of apoptotic activity, TUNEL assay was conducted using a DeadEnd Colorimetric TUNEL System in accordance with the manual protocol (Cat. No: G7360, Promega).
All statistical analysis was performed using SPSS version 17.0 Software (SPSS Inc., Chicago, IL). The comparisons between two or more groups were analyzed using two-tailed unpaired Student’s test or one-way ANOVA with Turkey’s post hoc test. A p-value was considered statistically significant when less than 0.05.